Treatment for dopaminergic disorders

ABSTRACT

The present invention provides systems, compositions and methods for treatment of dopaminergic disorders (e.g., Parkinson&#39;s disease) using the combination of a first compound that inhibits a voltage-gated calcium channel of the type Ca v 1.3 (e.g., a dihydropyridine such as isradipine), and a second compound that is monoamine oxidase inhibitor and/or is a nitric oxide synthase inhibitor (e.g., rasagiline or derivative thereof).

The present application is a continuation of U.S. patent applicationSer. No. 14/253,528, filed Apr. 15, 2014, now allowed, which claimspriority to U.S. Provisional Application Ser. No. 61/811,993, filed Apr.15, 2013, each of which is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to systems, compositions and methods fortreatment of dopaminergic disorders (e.g., Parkinson's disease) usingthe combination of a first compound that inhibits a voltage-gatedcalcium channel of the type Ca_(v)1.3 (e.g., a dihydropyridine such asisradipine), and a second compound that is monoamine oxidase inhibitorand/or is a nitric oxide synthase inhibitor (e.g., rasagiline orderivative thereof).

BACKGROUND OF THE INVENTION

Dopamine is a chemical that the body produces naturally in the brain. Inthe brain, dopamine functions as a neurotransmitter and is a criticalcomponent used by the brain to control bodily movements, thereforechanges in the level of dopamine in the brain can have devastatingresults. Augmentation in the levels of dopamine in the brain has beenassociated with conditions such as drug addition, psychiatric disorderslike schizophrenia, depression, Parkinson's Disease, andParkinsonian-like disorders.

Parkinson's Disease (PD) is a progressive disorder of the centralnervous system that affects over one million people in the United Statesalone and is associated with a loss in dopamine production in a specificarea of the brain. Approximately 40,000 Americans are diagnosed with PDevery year, and although PD is typically equated to be a diseaseafflicting older adults, more and more people are being diagnosed withthe disease before the reach the age of 50. It is a chronic andprogressive disease, meaning that the symptoms of PD grow worse and lastover time. Characteristic symptoms include a decrease in spontaneousmovements, gait difficulty, postural instability, rigidity and tremors.

As with a number of neurological diseases, the true cause of PD is notknown. However, research has shown that Parkinson's Disease occurs whena group of neuronal cells in the area of the brain called the substantianigra pars compacta (SNc), begin to malfunction and eventually dieleading to a decrease in levels of dopamine in the brain, which in turnleads to impaired motor control and coordination. There is currently noknown cure for PD. To date, treatment has been directed to increasingthe amount of dopamine in the brain by drug administration, or to moreinvasive surgical treatments such as targeted neuronal ablation and deepbrain stimulation. Unfortunately, all treatments suffer from drawbacks,some serious, which debilitate the patient and compromise the quality oflife.

What are needed are novel ways of understanding and studying these typesof dopamine related disorders and diseases, and novel ways of treatingthese types of disorders and diseases without disrupting normal neuronalfunctioning and compromising the quality of life of those afflicted.

SUMMARY OF THE INVENTION

The present invention relates to systems, compositions and methods fortreatment of dopaminergic disorders (e.g., Parkinson's disease) usingthe combination of a first compound that inhibits a voltage-gatedcalcium channel of the type Ca_(v)1.3, such as Ca_(v)1.3a (e.g., adihydropyridine such as isradipine), and a second compound that ismonoamine oxidase inhibitor and/or is a nitric oxide synthase inhibitor(e.g., rasagiline or derivative thereof).

In some embodiments, the present invention provides methods of treatmentfor dopaminergic disorders comprising: administering to a subject havinga dopaminergic disorder: i) a first compound that inhibits avoltage-gated calcium channel of the type Ca_(v)1.3 (e.g., Ca_(v)1.3a),and ii) a second compound that is monoamine oxidase inhibitor and/or isa nitric oxide synthase inhibitor.

In particular embodiments, the present invention provides compositionscomprising: i) a first compound that inhibits a voltage-gated calciumchannel of the type Ca_(v)1.3 (e.g., Ca_(v)1.3a), and ii) a secondcompound that is monoamine oxidase inhibitor and/or is a nitric oxidesynthase inhibitor.

In further embodiments, the present invention provides systems and kitscomprising: i) a first compound that inhibits a voltage-gated calciumchannel of the type Ca_(v)1.3 (e.g., Ca_(v)1.3a), and ii) a secondcompound that is monoamine oxidase inhibitor and/or is a nitric oxidesynthase inhibitor.

In certain embodiments, the first compound that modulates the activityand/or expression of voltage-gated calcium channels of the typeCa_(v)1.3 is a calcium channel blocker. In other embodiments, thecalcium channel blocker is a dihydropyridine calcium channel blocker. Infurther embodiments, the dihydropyridine calcium channel blocker isselected from a group consisting of nifedipine, nimopidine, isradipineor a compound shown in Pat. Pub. 20120046309 (see structure in paragraph7) or a compound described in Pat. Pub. 20120196883 (see structure inparagraphs 11-27), both of which are herein incorporated by reference intheir entirety. In further embodiments, the first compound thatmodulates the activity and/or expression of voltage-gated calciumchannels of the type Ca_(v)1.3 is a nucleic acid. In other embodiments,the nucleic acid is a small interfering RNA. In particular embodiments,the second compound is both a monoamine oxidase inhibitor and a nitricoxide synthase inhibitor. In other embodiments, the second compoundcomprises rasagiline or a rasagiline derivative. Rasagiline derivativesare known in the art. Examples include, but are not limited to,Ladostigil, M-30, TV3326 and TV3279.

The neurodegenerative disorder Parkinson's Disease is caused by thedeath of dopaminergic neurons in the substantia nigra pars compacta(SNc). The etiology of the disease is not understood, however mutationsin genes associated with mitochondria or protein folding are associatedwith human PD. These genes, however, are not expressed exclusively inthe dopaminergic SNc neurons and animals harboring these mutations donot necessarily develop, nor show signs, of the disease. The majority ofPD cases are not associated with any known genetic defect, and most ofthe current thinking suggests that exposure to environmental toxins,like rotenone, are responsible for these cases. Rotenone doesselectively kill SNc DA neurons in rats, but it is not clear why as itis a mitochondrial toxin that enters and affects all neurons. Otherdopaminergic disorders include mental disorders (e.g., schizophrenia,depression, drug addiction) and Parkinsonian-like disorders (e.g.,juvenile parkinsonism, Ramsey-Hunt paralysis syndrome).

Most medications used to treat Parkinson's Disease and otherdopaminergic system disorders, either mimic the effect of dopamine,increase dopamine levels, or extend the action of dopamine in the brain.The gold standard by which all treatments for PD are measured is theadministration of levodopa, which is a substance that is converted intodopamine in the brain. However, levodopa typically is administered aspart of a chemical cocktail, for example with carbidopa that preventsthe levodopa from being converted to dopamine in the bloodstream, and/orentacapone which extends the time levodopa is active in the brain. Thereare also a number of dopamine agonists which are administered, e.g.,bromocriptine, pergolide, pramipexole, and ropinirole. Additionaltreatments include chemicals that do not act directly on thedopaminergic system, but alternatively target another neurotransmitter,acetylcholine, which is in overabundance when the dopaminergic systemceases to create dopamine. The imbalance of acetylcholine relative todopamine levels causes additional physiological problems. Thosechemicals that target acetylcholine, anticholinergics, includetrihexyphenidyl, benzotropine mesylate, MAO and COMT inhibitors such asselegiline, deprenyl, entacapone (previously described), and tolcaponeare sometimes administered to help prolong the efficacy of the levodopaby slowing its breakdown in the brain, thereby helping to provide a morestable, constant supply of levodopa. Unfortunately, all of these drugsalone or in combination are associated with a variety of side effects,and potential drug interaction problems, which make their usage lessthan desirable. Other extreme, more invasive, measures to correctdopaminergic disorders include the targeted destruction of afflictedneuronal cells in the affected area of the brain, and deep brainstimulus. Both of these invasive procedures carry the risk of stroke andother operative complications.

It is contemplated that the death of dopaminergic neurons can beattributed to their reliance upon calcium channels. The calcium channelsfound in dopaminergic neurons, voltage-gated L-type calcium channels ofwhich Ca_(v)1.3a is one, autonomously generate influxes of calcium intodopaminergic neurons. The intracellular calcium loading caused by thisprocess, also referred to as pacemaking, synergizes with the stresscreated by the factors that potentially cause dopaminergic disorders(e.g., environmental toxins, genetic mutations, and the like) therebyinducing preferential death of the dopaminergic neurons and the onset ofdisease. Young ‘juvenile’ neurons don't depend on calcium to the extentthat aged neurons do, i.e. as neurons age they require more calcium. Itis contemplated that by reducing the calcium loading during pacemaking,the neurons will be forced to convert to a more ‘juvenile’ form ofpacemaking (an altered pacemaking mechanism), one that relies uponbetter tolerated ions such as sodium. This can be achieved by modulating(e.g., disrupting) pharmacologically or genetically the Ca_(v)1.3channels in the SNc dopaminergic neurons.

Current pharmacological therapies depend upon continued viability ofdopaminergic neurons affected by the diseases. However, as the diseaseprogresses and these neurons die, current pharmacological approachesfail to provide symptomatic relief. None of the current therapies slowprogression of the disease or the death or dopaminergic neurons. Bydirectly targeting the Ca_(v)1.3 channels (e.g., Ca_(v)1.3a channels) asdescribed in the present invention, damaged cells are protected fromfurther degradation and eventual death. Additionally, pre-treatment ofsubjects, by targeting Ca_(v)1.3 (e.g., Ca_(v)1.3a) channels using themethods and compositions of the present invention, who may bepre-disposed to developing a dopaminergic disorder (e.g., viaenvironmental toxin exposure, genetic disposition) provides apreventative therapy for these types of dopaminergic disorders.

There is a real need for novel ways to treat patients with dopaminergicdisorders which bypass the dopaminergic system thereby allowing analternative treatment for those afflicted with these types of disorders.It is contemplated that alternative treatments would bypass the sideeffects associated with present treatment regimes. Therefore, thepresent invention relates to methods and compositions for modulatingcalcium channels. In particular, the present invention provides methods,compositions, and kits for modulating (e.g., disrupting) Ca_(v)1.3(e.g., Ca_(v)1.3a) calcium channels. The methods, compositions, and kitsdescribed herein can be utilized to provide novel ways of treating andstudying dopaminergic related disorders.

In one embodiment, the method of the present invention is a method oftreatment for dopaminergic disorders comprising administering a compoundthat inhibits a voltage-gated calcium channel of the type Ca_(v)1.3 to asubject having a dopaminergic disorder. In some embodiments, the methodcomprises the administration of a calcium channel blocker, preferably adihydropyridine calcium channel blocker. In some embodiments, thedihydropyridine calcium channel blocker comprises nifedipine,nimodipine, and/or isradipine.

In one embodiment, the method of the present invention is a method toidentify compounds that inhibit activity and/or expression of avoltage-gated calcium channel of the type Ca_(v)1.3 comprising providinga compound suspected of inhibiting the expression or activity of aCa_(v)1.3 calcium channel, applying the compound to a sample whichcontains Ca_(v)1.3 calcium channels, and determining whether or not thecompound has affected the activity and/or expression of Ca_(v)1.3calcium channels. In some embodiments, the compounds to be testedcomprise nucleic acids (e.g., siRNA), and small molecules, antibodies,peptides, proteins, and the like.

In one embodiment, the method of the present invention is a method ofco-therapy treatment for dopaminergic disorders comprising providing acompound that inhibits the activity and/or expression of a voltage-gatedcalcium channel of the type Ca_(v)1.3 in conjunction with an additionaltherapeutic compound that is useful in treating dopaminergic disorders,and administering the combination to a subject suspected of having adopaminergic disorder. In some embodiments, the additional therapeuticcompound comprises a dihydropyridine calcium channel blocker and/or anucleic acid. In some embodiments, the additional therapeutic agentcomprises levodopa, carbidopa, entacapone, apomorphine hydrochloride,bromocriptine, pergolide, pramipexole, ropinirole, benzotropinemesylate, trihexyphenidyl HCl), selegiline, rasagiline, tolcapone,amantadine, riluzole, and/or L-dopa ethyl ether.

In one embodiment, the composition of the present invention comprises acompound that inhibits the activity and/or expression of voltage-gatedcalcium channels of the type Ca_(v)1.3 and is useful in treatingdopaminergic disorders. In some embodiments, the compound that inhibitsa Ca_(v)1.3a calcium channel comprises a calcium channel blocker and/ora nucleic acid. In some embodiments, the calcium channel blockercomprises a dihydropyridine calcium channel blocker. In someembodiments, the dihydropyridine calcium channel blocker comprisesnifedipine, nimodipine, and/or isradipine. In some embodiments, acompound that inhibits the activity and/or expression of voltage-gatedcalcium channel of the type Ca_(v)1.3 is a nucleic acid, such as a smallinterfering RNA. In some embodiments, the compound that inhibits theactivity and/or expression of voltage-gated calcium channel of the typeCa_(v)1.3 is further combined with an additional therapeutic agentcomprising levodopa, carbidopa, entacapone, apomorphine hydrochloride,bromocriptine, pergolide, pramipexole, ropinirole, benzotropinemesylate, trihexyphenidyl HCl), selegiline, rasagiline, tolcapone,amantadine, riluzole, and/or L-dopa ethyl ether.

In certain embodiments, provided herein are methods of treatment fordopaminergic disorders comprising: administering to a subject having adopaminergic disorder: i) a first compound that inhibits a voltage-gatedcalcium channel of the type Ca_(v)1.3, and ii) a second compound that ismonoamine oxidase inhibitor and/or is a nitric oxide synthase inhibitor.In certain embodiments, the first compound comprises a dihydropyridinecalcium channel blocker. In further embodiments, the first compoundcomprises isradipine (or an isradipine derivative) and the secondcompound comprises rasagiline or a rasagiline derivative. I certainembodiments, the first compound is a compound shown in Pat. Pub.20120046309 (see structure in paragraph 7; herein incorporated byreference) or a compound described in Pat. Pub. 20120196883 (seestructure in paragraphs 11-27; herein incorporated by reference), bothreferences of which are herein incorporated by reference in theirentirety.

In particular embodiments, the administering the first and secondcompounds to the subject is repeated on multiple days (e.g., on 2 days,3 days . . . 10 days . . . 15 days, etc.). In some embodiments, about 10mg of the first compound (e.g., isradipine) is administered on one ormore days. In some embodiments, no more than 9 mg of the first compoundis administered to the subject on any given day (e.g., not more than 9,8, 7, 6, 5, 4, or 3 mgs are administered on a single day). In furtherembodiments, the first compound comprises isradipine. In otherembodiments, the second compound comprises rasagiline or a rasagilinederivative. In other embodiments, the first compound comprisesisradipine, wherein the second compound comprises rasagiline or arasagiline derivative, wherein between about 0.5 mg and about 5.0 mg ofthe isradipine is administered per day (e.g., 0.5 mg . . . 1.0 mg . . .2.0 mg . . . 3.0 mg . . . 4.0 mg . . . or 5.0 mg), and wherein about 0.5mg and about 2.0 mg of the rasagiline or rasagiline derivative isadministered per day (e.g., 0.5 mg . . . 0.9 mg . . . 1.3 mg . . . 1.6mg . . . 1.9 mg, 2.0 mg . . . 2.2 mg). In other embodiments, the firstand/or second compound is in a controlled release formulation.

In some embodiments, provided herein are compositions comprising: i) afirst compound that inhibits a voltage-gated calcium channel of the typeCa_(v)1.3a, and ii) a second compound that is monoamine oxidaseinhibitor and/or is a nitric oxide synthase inhibitor. In certainembodiments, the first compound comprises a dihydropyridine calciumchannel blocker. In other embodiments, the first compound comprisesisradipine (or an isradipine derivative) and the second compoundcomprises rasagiline or a rasagiline derivative. In further embodiments,the first composition contains no more than 9 mg of the first compound(e.g., not more than 9, 8, 7, 6, 5 mgs, 4 mgs or 3 mgs). In furtherembodiments, the first compound comprises isradipine. In otherembodiments, the second compound comprises rasagiline or a rasagilinederivative.

In certain embodiments, the first compound comprises isradipine, whereinthe second compound comprises rasagiline or a rasagiline derivative,wherein the composition contains between about 0.5 mg and about 5.0 mg(e.g., 0.5 mg . . . 1.0 mg . . . 2.0 mg . . . 3.0 mg . . . 4.0 mg . . .or 5.0 mg) of the isradipine and between about 0.5 mg and about 2.0 mg(e.g., 0.5 mg . . . 0.9 mg . . . 1.3 mg . . . 1.6 mg . . . 1.9 mg, 2.0mg . . . 2.2 mg) of the rasagiline or rasagiline derivative.

In certain embodiments, provided herein are compositions comprising thecombination of isradipine (or isradipine derivative) and rasagiline (orrasagiline derivative), where the isradipine (or derivative thereof) ispresent at about 1 mg, 2 mg, 3 mg, 4 mg, or about 5 mg, and therasagiline or derivative thereof is present at about 0.5 mg, 1 mg, 1.5mg, or about 2.0 mgs. Any and all combinations of such dosages ofisradipine and rasagiline are contemplated. For example, about 5 mg ofisradipine and about 2 mg of rasagiline in a composition (e.g., in apill or capsule), or 4-6 mg of isradipine and about 1-3 mg ofrasagiline. In certain embodiments, the isradipine or derivative thereofis present in the composition in a controlled release formulation.

In particular embodiments, provided herein are kits and systemscomprising: i) a first composition comprising a first compound thatinhibits a voltage-gated calcium channel of the type Ca_(v)1.3, and ii)a second composition comprising a second compound that is monoamineoxidase inhibitor and/or is a nitric oxide synthase inhibitor. Inparticular embodiments, the first compound comprises isradipine (orderivative thereof) and the second compound comprises rasagiline or arasagiline derivative. In other embodiments, the first compositioncontains no more than 9 mg of the first compound (e.g., not more than 9,8, 7, 6, 5 mgs, 4 mgs or 3 mgs). In some embodiments, the kit furthercomprises a container (e.g., box or other package) and the first andsecond compositions are within the container. In particular embodiments,the first composition is in a first container (e.g., pill bottle) andthe second composition is in a second container (e.g., pill bottle). Inparticular embodiments, the first and second compositions are in pill orcapsule form.

In certain embodiments, provided herein are methods of treatingParkinson's disease with high levels of rasagiline or a derivativethereof (e.g., treatment at or above 3 mg/kg of subject per day formultiple days). Such as 3, 4, 5, 6 . . . 10 or 15 mg/kg of patient perday.

DESCRIPTION OF THE FIGURES

FIG. 1 shows inhibition of L-type calcium channels with thedihydropyridine calcium channel antagonist nimodipine, which inhibitsautonomous pacemaking of dopaminergic neurons in a tissue slice from anadult.

FIG. 2 shows dopaminergic neurons co-express Ca_(v)1.3a (long splicevariant) and Ca_(v)1.3b (short splice variant) of the Ca_(v)1.3 gene.

FIG. 3 demonstrates that pacemaking is accompanied by large fluctuationsin the concentration of dendritic calcium in SNc dopaminergic neurons.

FIG. 4 shows that the altered pacemaking mechanism can be elicited in anadult SNc dopaminergic neuron by inhibiting Ca_(v)1.3 channels for abrief period. Recordings are shown from a SNc dopaminergic neuronbefore, during, and after application with the dihydropyridine calciumchannel blocker isradipine.

FIG. 5 demonstrates that knocking out Ca_(v)1.3 channels confersresistance to the pesticide rotenone, a member of a class ofenvironmental agents thought to contribute to idiopathic Parkinson'sDisease. Sections from wt and Ca_(v)1.3 knock-out mouse brains arestained for tyrosine hydroxylase after application of 100 nM rotenoneand 10 μM glibenclamide.

FIG. 6 shows that rasagiline does not alter pacemaking rate orintracellular calcium oscillations associated with pacemaking. Left:image of an SNc DA neuron in an ex vivo brain slice with an annulusdrawn to show the area from which dendritic calcium measurements weremade. Middle: Whole cell voltage measurements from an SNc DA neuron in acontrol slice; bottom are calcium oscillations associated withpacemaking made using the indicator Fluo4 and 2PLSM. Right: Similarmeasurements from a brain slice taken following rasagiline treatment.

FIGS. 7A-D show that mitochondrial oxidant stress is diminished bychronic isradipine or chronic rasagiline treatment. FIG. 7A:Photomicrograph of mesencephalon of BAC-mito-roGFP mouse showingtransgene expression in SNc and VTA DA neurons. Bottom: image of asingle neuron. FIG. 7B: Redox measurements in ex vivo brain slices werecalibrated by application of strong reducing (DTT) and oxidizing agents(aldrithiol) at the end of each experiment. FIG. 7C: Representativecontrol and acute rasagiline-induced responses. FIG. 7D: Box plotssummarizing mitochondrial oxidant stress measurements from mice givenisradipine or rasagiline acutely or chronically (1 week) with s.c.osmotic minipumps. N>4 in each group.

FIG. 8 shows that the effects of chronic administration of isradipine orrasagiline were dose-dependent. Panel a: Box plots summarizingmitochondrial oxidant stress measurements in SNc DA neurons in ex vivobrain slices from mice given saline or isradipine. Acute treatment atthe time of measurement (1 μM); other drugs were given for 1 week vias.c. osmotic minipump. Panel b: As in a, but with rasagiline. Acuterasagiline was given at X μM. N>4 in each group.

FIG. 9 shows that the combination of low dose isradipine and rasagilineis more effective than either alone. Panel a: Mouse with two s.c.osmotic minipumps; drugs were given for 1 week prior to sacrifice. Panelb: Box plots summarizing mitochondrial oxidant stress measurements inSNc DA neurons in ex vivo brain slices from mice given drugs alone or incombination. N>4 in each group.

DEFINITIONS

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include tissuesand blood products, such as plasma, serum and the like. Such examplesare not however to be construed as limiting the sample types applicableto the present invention.

As used herein, the term “peptide” refers to a compound comprising fromtwo or more amino acid residues wherein the amino group of one aminoacid is linked to the carboxyl group of another amino acid by a peptidebond. A peptide can be, for example, derived or removed from a nativeprotein by enzymatic or chemical cleavage, or can be prepared usingconventional peptide synthesis techniques (e.g. solid phase synthesis)or molecular biology techniques (see Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989)).

As used herein, the term “peptidomimetic” refers to molecules which arenot polypeptides, but which mimic aspects of their structures. Forexample, polysaccharides can be prepared that have the same functionalgroups as peptides. A peptidomimetic comprises at least two components,the binding moiety or moieties, and the backbone or supportingstructure.

As used herein, the term “antibody” encompasses both monoclonal andpolyclonal full length antibodies and functional fragments thereof (e.g.maintenance of binding to target molecule). Antibodies can include thosethat are chimeric, humanized, primatized, veneered or single chainantibodies.

As used herein, the term “dopaminergic disorder” refers to diseases andconditions associated with aberrant dopamine production. Dopaminergicdisorders include, but are not limited to, Parkinson's Disease, andParkinsonian-like disorders such as juvenile parkinsonism andRamsey-Hunt paralysis syndrome.

As used herein, the term “effective amount” of a therapeutic compound(e.g. agent, compound, or drug) is an amount sufficient to achieve adesired therapeutic and/or prophylactic effect, such as to inhibitneuronal cell death, or to alleviate behavioral disorders associatedwith a dopaminergic disorder.

As used herein, the terms “agent”, “compound” or “drug” are used todenote a compound or mixture of chemical compounds, a biologicalmacromolecule such as an antibody, a nucleic acid, or an extract madefrom biological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues that are suspected of havingtherapeutic properties. The compound, agent or drug may be purified,substantially purified or partially purified.

As used herein, the term “fragment” when in reference to a protein (e.g.“a fragment of a given protein”) refers to portions of that protein. Thefragments may range in size from two amino acid residues to the entireamino acid sequence minus one amino acid. In one embodiment, the presentinvention contemplates “functional fragments” of a protein. Suchfragments are “functional” if they can bind with their intended targetprotein (e.g. the functional fragment may lack the activity of the fulllength protein, but binding between the functional fragment and thetarget protein is maintained).

As used herein, the term “antagonist” refers to molecules or compounds(either native or synthetic) that inhibit the action of a compound(e.g., receptor channel, ion channel, etc.). Antagonists may or may notbe homologous to these compounds in respect to conformation, charge orother characteristics. Thus, antagonists may be recognized by the sameor different receptors that are recognized by an agonist. Antagonistsmay have allosteric effects that prevent the action of an agonist. Or,antagonists may prevent the function of the agonist.

As used herein, the term “therapeutically effective amount” refers tothat amount of a composition which results in amelioration of symptomsor a prolongation of survival in a patient. A therapeutically relevantamount relieves to some extent one or more symptoms of a disease orcondition, or returns to normal, either partially or completely, one ormore physiological or biochemical parameters associated with a diseaseor condition.

As used herein, the term “subject” refers to any biological entity thatcan be used for experimental work. For example, a “subject” can be amammal such as a mouse, rat, pig, dog, non-human primate. Preferably thesubject is a human primate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides systems, compositions and methods fortreatment of dopaminergic disorders (e.g., Parkinson's disease) usingthe combination of a first compound that inhibits a voltage-gatedcalcium channel of the type Ca_(v)1.3 (e.g., a dihydropyridine such asisradipine), and a second compound that is monoamine oxidase inhibitorand/or is a nitric oxide synthase inhibitor (e.g., rasagiline orderivative thereof).

In certain embodiments, combining the second compound (monoamine oxidaseinhibitor and/or is a nitric oxide synthase inhibitor) with the firstcompound (compound that inhibits a voltage-gated calcium channel of thetype Ca_(v)1.3) allows one to avoid side effects associated with highdoses of the first compound as the presence of the second compoundallows the first compound to be administered at lower doses. Forexample, while a typical dose of isradipine is 10 mg, combiningisradipine with rasagiline (e.g., 1-2 mg per day) allows one to use lessisradipine (e.g., 5 mg) and still achieve the same therapeutic effect(e.g., in treating Parkinson's disease), thereby avoiding some of theside effects associated with 10 mg per days of isradipine.

Neurons express multiple types of voltage-gated calcium (Ca²⁺) channels(Ca_(v)). Neuronal N-type and P/Q type Ca²⁺ channels have been shown tomediate Ca²⁺ influx that triggers release of neurotransmitters. NeuronalL-type Ca²⁺ channels do not trigger such a release even though theystill play a critical role in Ca²⁺ influx in neurons. There are twodistinctive subtypes of neuronal L-type Ca²⁺ channels; Ca_(v) 1.2 andCa_(v) 1.3. The Ca_(v) 1.3 channel subtype is further alternativelyspiced at its C-terminus to yield two forms of Ca_(v) 1.3; Ca_(v) 1.3a(long splice variant) and Ca_(v) 1.3b (short splice variant). The longsplice variant Ca_(v) 1.3a contains SH3 and class I PDZ binding domainsthat selectively bind to Shank scaffolding proteins, the combination ofwhich has been found targeted to glutamatergic synapses in striatalmedium spiny neurons (MSN). This scaffolding interaction enablesmodulation of the channel by the two dopamine receptor types, D₁dopaminergic receptor which is expressed by MSNs projecting to thesubstantia nigra, and D₂ dopaminergic receptor which is expressed byMSNs projecting to the globus pallidus. Additionally, dopaminergicreceptors have been shown to suppress Ca²⁺ influx through L-type Ca_(v)channels in rodent striated MSNs.

Calcium loading of neurons is an autonomous process. It is contemplatedthat this process is potentially tied to the deafferentation of neuronsthat leads to dopaminergic disorders like Parkinson's Disease. It iscontemplated that the deafferentation of neurons is a consequence ofaltered modulation of the synaptically targeted Ca_(v) 1.3a calciumchannels by the D₂ receptor and that partial disruption of thesechannels prevents the structural adaptation following dopaminedepletion. Ca_(v) 1.3a channels control synaptic plasticity andconnectivity of striatal MSNs. D₂ receptor activation reduces Ca_(v) 1.3channel open probability whereas D₁ receptor activation increaseschannel open probability, or doesn't change it significantly. Geneticdeletion of Ca_(v) 1.3a channels results in a dramatic increase inglutamatergic synapsis and spines in MSNs suggesting that calcium fluxthrough this channel is a critical negative regulator of synapsestability. The loss of dopamine receptors seen in PD leads to a loss ofD₂ receptors, and consequently to an increase in the Ca_(v) 1.3 channelopen probability, followed by increases in intracellular calcium intospine heads of stratopallidal neurons, and the eventual loss ofglutamatergic synapses in these neurons. The synaptic loss effectivelycauses deafferentation of the striatopallidal neurons, preventing themfrom fulfilling their role in motor control. This loss is contemplatedto be a major factor contributing to the pathophysiology underlyingdopaminergic disorders such as PD.

Disruption of Ca_(v) 1.3 channels prevents this deafferentationfollowing the loss of dopamine, and the targeting of Ca_(v) 1.3 channelsdoes not depend upon retention of dopaminergic neurons nor is itaffected by alteration in signaling pathways triggered by diseaseprogression. For example, when a Ca_(v) 1.3 knock-out mouse was treatedwith rotenone, a pesticide suggested to cause idiopathic PD, the SNcneurons looked indistinguishable from untreated tissue whereas thewild-type (wt) mice exhibited SNc deafferentation (FIG. 5). As will belater demonstrated, these calcium channel knock-out mice demonstrate noill side affects due to the deletion based upon behavioral studies(Example 5). The disruption of the Ca_(v) 1.3 calcium channels, eitherthrough pharmacological blockade or channel deletion, lead to an alteredpacemaking mechanism where SNc dopaminergic neurons switch to sodiumchannel dependent pacemakers without changing the pacemaking rate. Thealtered pacemaking in dopaminergic neurons following Ca_(v)1.3 deletiondoes not depend upon alterations in the sodium channel expression orfunction, rather it depends upon modulation of endogenously expressedhyperpolarization activated cation channels, which explains why normalneurons are capable of compensating when disruption of Ca_(v) 1.3calcium channels occurs (e.g. through pharmacological blockade).

Therefore, targeted pharmacological (e.g., antagonists, drugs, agents,and the like) or genetic disruption (e.g., siRNA, and the like) ofCa_(v) 1.3a is a novel therapeutic strategy that does not depend upon anintact dopaminergic innervation of the striatum as do current treatmentstrategies. It is contemplated that the prevention of deafferentationand subsequent dopamine loss found in some dopaminergic disorders willameliorate the symptoms of the disease, and the specific targeting of acellular protein will significantly decrease the side affects of currentdopamine replacement strategies. The combination therapy of disruptingthe Ca_(v) 1.3a and the administration of existing therapies (e.g.,levodopa, carbidopa, etc.) will broaden the window for treatment suchthat both cellular deafferentation will be averted and dopamine levelswill be ameliorated leading to a better quality of life for thoseafflicted with dopaminergic disorders.

In one embodiment, the method of the invention comprises the modulationof L-type calcium channels found in dopaminergic neurons. In someembodiments, the modulation of L-type calcium channels involvesmodulating Ca_(v)1.3 channels found in dopaminergic neurons. In someembodiments, modulation of Ca_(v) 1.3 channels further involvesmodulating a subtype of the Ca_(v) 1.3 channel, the Ca_(v) 1.3a channel.In some embodiments, the modulation of Ca_(v) 1.3a channels provides atreatment for Parkinson's Disease and other dopaminergic disorders ofthe basal ganglia (e.g. parkinsonian-type disorders, and the like). Insome embodiments, the present invention provides methods, compositions,and kits for use in the modulation of Ca_(v) 1.3a channels. It iscontemplated that Ca_(v) 1.3a channels may be modulated using anymethods including, but not limited to, biochemical, genetic, and othermethods known in the art.

Some embodiments of the present invention relate to therapeutic methodsand compositions for treating a subject having a dopaminergic disorderor healthy subjects. In some embodiments, the method of treatmentcomprises the administration of an antagonist, agent, compound, or drugto a subject having a dopaminergic disorder or to healthy subjects(e.g., prophylactic treatment), such as subjects with a predispositionto, or risk of, acquiring a dopaminergic disorder (e.g., exposure toenvironmental toxins such as rotenone, genetic disposition, etc.). Insome embodiments, the antagonist physically interacts with the Ca_(v)1.3a calcium channel, or the antagonist blocks production of the Ca_(v)1.3a calcium channel, e.g. by inhibiting translation of the receptorgene into a protein product. In one embodiment, the antagonist is asiRNA that inhibits translation of the Ca_(v) 1.3a calcium channel gene.Another embodiment comprises the administration of a calcium channelblocker(s) to a subject having a dopaminergic disorder. One embodimentcomprises the administration of an antibody, antibody fragment, orpeptide that would block the calcium channel. One embodiment of atherapeutic method of treatment comprises the administration of acalcium channel blocker from the dihydropyridine class of compounds. Forexample, dihydropyridine calcium channel blockers include, but are notlimited to isopropyl 2-methoxyethyl1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylate(e.g., nimopidine, Nimotop® Bayer Corporation NDA#18-869),3,5-pyridinedicarboxylic acid,4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-, methyl 1-methylethylester (e.g., isradipine, DynaCirc® Sandoz Pharmaceuticals NDA#19-546),and dimethyl1,4-hydro-2,6-dimethyl-4-(o-nitrophenyl)-3,5-pyridinecarboxylate (e.g.,nifedipine Biovail Laboratories, Inc. ANDA#75-269, Adalat® BayerCorporation, U.S. Pat. Nos. 4,892,741 and 5,264,446, ELANPharmaceuticals ANDA#75-128, Mylan Pharmaceuticals ANDA#75-108) andderivatives thereof (all cited references are incorporated herein byreference). It is further contemplated that dihydropyridine analogs areadministered (e.g., nilvadipine, mesudipine, and the like). A preferredembodiment comprises the administration of a dihydropyridine calciumchannel blocker or analog thereof to a subject suffering from adopaminergic disorder. Some embodiments comprises the administration ofisradipine to a subject suffering from a dopaminergic disorder.Typically, an effective amount of a dihydropyridine calcium channelblocker can range from about 0.01 mg per day to greater than 2000 mg perday for an adult, although other doses are contemplated. In someembodiments, the dosage ranges from about 1 mg per day to about 120 mgper day. In other embodiments, the dosage ranges from about 2 mg per dayto about 90 mg per day (e.g., about 5 mg or about 10 mg per day). It iscontemplated that the dosage is administered, for example, continuouslyon a daily, weekly, monthly, or yearly basis. Dihydropyridine calciumchannel blockers are well tolerated by human subjects, and are used intherapeutic regimens for other diseases and disorders (e.g.,cardiovascular conditions, etc.).

In some embodiments the present invention provides methods of storageand administration of the antagonist, agent, compound, or drug in asuitable environment (e.g. buffer system, adjuvants, etc.) in order tomaintain the efficacy and potency of the agent, compound, or drug suchthat its usefulness in a method of treatment of a dopaminergic disorderis maximized. For example, protein agents, chemicals or nucleic acidsbenefit from a storage environment free of proteinases and other enzymesor compounds that could cause degradation of the protein, chemical, ornucleic acid.

A preferred embodiment is contemplated where the antagonist, agent,compound, or drug is administered to the individual as part of apharmaceutical or physiological composition for treating dopaminergicdisorders. Such a composition can comprise an antagonist and aphysiologically acceptable carrier. Pharmaceutical compositions forco-therapy can further comprise one or more additional therapeuticagents. The formulation of a pharmaceutical composition can varyaccording to the route of administration selected (e.g., solution,emulsion, capsule). Suitable pharmaceutical carriers can contain inertingredients that do not interact with the antagonist of Ca_(v) 1.3afunction and/or additional therapeutic agent. Standard pharmaceuticalformulation techniques can be employed, such as those described inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa. Suitable physiological carriers for parenteral administrationinclude, for example, sterile water, physiological saline,bacteriostatic saline (saline containing about 0.9% benzyl alcohol),phosphate-buffered saline, Hank's solution, Ringer's-lactate and thelike. Methods for encapsulating compositions (such as in a coating ofhard gelatin or cyclodextran) are known in the art (Baker, et al,“Controlled Release of Biological Active Agents”, John Wiley and Sons,1986). The particular co-therapeutic agent selected for administrationwith an antagonist of Ca_(v) 1.3a calcium channel will depend on thetype and severity of the dopaminergic disorder being treated as well asthe characteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs.

In some embodiments the therapeutic agent is administered by anysuitable route, including, for example, orally (e.g., in capsules,suspensions or tablets) or by parenteral administration. Parenteraladministration can include, for example, intramuscular, intravenous,intraarticular, intrathecal, subcutaneous, or intraperitonealadministration. The therapeutic agent (e.g., Ca_(v) 1.3 antagonist,nucleic acid, additional therapeutic agent) can also be administeredtransdermally, topically, by inhalation (e.g., intrabronchial,intranasal, oral inhalation or intranasal drops) or rectally.Administration can be local or systemic as indicated. The preferred modeof administration can vary depending upon the particular agent chosen,however, oral or parenteral administration is generally preferred. Atimed-release, subcutaneous mode of administration is also contemplated.For example, a therapeutic agent is inserted under the skin either byinjection, and/or by placing a solid support that has been previouslyimpregnated or which contains (e.g., a capsule) the therapeutic agent,under the skin. An effective amount of the therapeutic agent is thenreleased over time (e.g., days, weeks, months, and the like) such thatthe subject is not required to have a therapeutic agent administered ona daily basis. In some circumstances where high brain levels of theantagonist are desired, intrathecal injection or direct administrationinto the brain tissue is contemplated.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. In other embodiments, the activeingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers and are made bycompressing or molding the respective agents. In some embodiments,compressed tablets are prepared by compressing in a suitable machine theactive ingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent. Molded tablets are made by molding in a suitablemachine a mixture of the powdered compound (e.g., active ingredient)moistened with an inert liquid diluent. Tablets may optionally be coatedor scored and may be formulated so as to provide slow or controlledrelease of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in parts of the gut other than thestomach.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. In some embodiments, the formulations arepresented/formulated in unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies. Still other formulations optionally include food additives(suitable sweeteners, flavorings, colorings, etc.), phytonutrients(e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, andother acceptable compositions (e.g., conjugated linoelic acid),extenders, and stabilizers, etc.

When co-administration of an antagonistic therapeutic agent (e.g.,Ca_(v) 1.3 antagonist, nucleic acid, additional therapeutic agent) andan additional therapeutic agent (e.g., rasagiline) is indicated ordesired for treating a dopaminergic disorder, the antagonistictherapeutic agent can be administered prior to, concurrently with, orsubsequent to administration of the additional therapeutic agent. Whenthe antagonistic therapeutic agent and the additional therapeutic agentare administered at different times, they are preferably administeredwithin a suitable time period to provide substantial overlap of thepharmacological activity of the agents. The treating physician will beable to determine the appropriate timing for co-administration ofantagonistic therapeutic agents and an additional therapeutic agent.Examples of additional therapeutic agents that are administered priorto, concurrently, or subsequent to administration of the antagonistictherapeutic agent (e.g., Ca_(v) 1.3 antagonist, nucleic acid, additionaltherapeutic agent) include, but are not limited to, levodopa, carbidopa,entacapone (e.g., Comtan®), levodopa with carbidopa (e.g., Sinemet®),controlled released levodopa with carbidopa (e.g., Sinemet CR®),levodopa with carbidopa and entacapone (e.g., Stalevo®), apomorphinehydrochloride (e.g., APOKYN™), bromocriptine (e.g., Parlodel®),pergolide (e.g., Permax®), pramipexole (e.g., Mirapex®), ropinirole(e.g., Requip®), benzotropine mesylate (e.g., Cogentin®),trihexyphenidyl HCl (e.g., Artane®), selegiline (e.g., Eldepryl®,Carbex®), tolcapone (e.g., Tasmar®), amantadine (e.g., Symmetrel®),riluzole, L-dopa ethyl ether, potassium channel blockers, benzoxazinesand its derivatives, chloroquine and its derivatives, cGMP PDEinhibitors, Coenzyme Q10, Vitamin E, and Vitamin C or any compounds orderivatives thereof as referenced in U.S. Pat. Nos. 5,853,385,6,920,359, 6,911,475, 6,812,228, 6,756,056, 6,670,378, 6,653,325,6,620,792, 6,620,415, 6,608,064, 6,515,131, 6,514,999, 6,506,729,6,506,378, 6,492,371, 6,417,210, 6,417,177, 6,387,936, 6,330,888,6,309,634, 6,306,403, 6,300,329, 6,277,887, 6,200,607, 6,197,339,6,166,081, 6,106,839, 6,106,491, 5,980,914, 5,965,571, 5,948,806,5,863,925, 5,756,550, 5,702,700, 5,686,423, 5,677,344, 5,674,885,5,658,900, 5,650,443, 5,607,969, 5,597,309, 5,587,378, 5,565,460, and5,547,969 incorporated by reference herein in their entireties.

In other embodiments, the present invention provides methods ofscreening compounds for their ability to inhibit Cav1.3a channels. Insome embodiments, the present invention provides drug-screening assays(e.g., to screen for drugs effective in treating dopaminergicdisorders). For example, the present invention contemplates methods ofscreening for compounds that modulate (e.g., decrease) the expressionlevel or activity of a Ca_(v) 1.3a calcium channel. In one embodiment,the expression level of a Ca_(v) 1.3a calcium channel or its activity isdetected in vitro in a subject upon administration of a candidatecompound. The presence of a Ca_(v) 1.3a calcium channel or its continuedor increased activity is indicative of a candidate compound that is notpreventing a dopaminergic disorder. In some embodiments, the expressionlevel or activity of a Ca_(v) 1.3a calcium channel is detected using anin vitro assay, for example, an enzyme-linked immunosorbent assay, orother assays which utilize a labeled (e.g., fluorescent, luminescent,colorimetric, radioactive) compound for detection of a protein productor channel activity. In other embodiments, the expression level ofCa_(v) 1.3a calcium channels can be detected using RT-PCR techniques asdescribed herein. Antagonists of Ca_(v) 1.3a calcium channels can beidentified, for example, by screening libraries or collections ofmolecules, such as the Chemical Repository of the National CancerInstitute, as described herein or using other suitable methods.Antagonists thus identified find use in the therapeutic methodsdescribed herein. Another source for identifying potential antagonistsof Ca_(v) 1.3a calcium channels are combinatorial libraries, which cancomprise many structurally distinct molecular species. Combinatoriallibraries can be used to identify compounds or to optimize a previouslyidentified compound. Such libraries can be manufactured by well-knownmethods of combinatorial chemistry and can be screened by suitablemethods, such as those described in Molecular Cloning: A LaboratoryManual Sambrook J et al Eds, Cold Harbor Spring Laboratory Press.

In some embodiments, drug screening assays are performed in animals. Anysuitable animal may be used including, but not limited to, baboons,rhesus or other monkeys, mice, or rats. Animal models of dopaminergicdisorders are generated, and the effects of candidate drugs on theanimals are measured. In preferred embodiments, dopaminergic disordersin the animals are measured by detecting levels of Ca_(v) 1.3a calciumchannels in the affected tissues (e.g. SNc, MSN, other neuronal tissues)of the animals. The expression level or activity of related Ca_(v) 1.3acalcium channels may be detected using any suitable method, including,but not limited to, those disclosed herein (e.g., tissue analysis,nucleic acid analysis, behavioral analysis, etc.).

The present invention is not limited by the nature of the antagonistused in the therapeutic or screening methods of the invention. In oneembodiment, the antagonist is a nucleic acid such as a small interferingRNA (siRNA), which inhibits the translation of the mRNA encoding theCa_(v) 1.3a channel. Creation and use of siRNA is well known by thoseskilled in the art. For example, specialized software such as BLOCK-iT™RNAi Designer (Invitrogen Corporation) designs targeted RNAi moleculesto user defined sequences, and reference manuals (e.g., Hannon G J ed.,2003, RNAi: A Guide to Gene Silencing, Cold Spring Harbor LaboratoryPress, p. 436.) to RNA interference applications are readily available,and are incorporated by reference herein in their entirety.

In some embodiments, an antagonist of Ca_(v) 1.3a calcium channels doesnot significantly inhibit the function of other neuronal calciumchannels (e.g., Ca_(v) 1.2, Ca_(v) 1.3b N-type, P/Q-type calciumchannels, and the like). Such Ca_(v) 1.3a-specific antagonists can beidentified by suitable methods, such as by suitable modification of themethods described herein. For example, cells which do not express Ca_(v)1.3a but do express one or more other neuronal calcium channels (e.g.,Ca_(v) 1.2, Ca_(v) 1.3b N-type, P/Q-type calcium channels, and the like)can be screen for channel specificity. Such cells or cellular fractions(e.g., membranes) obtained from such cells can be used in a suitablebinding or activity assay. For example, if a cell lacks Ca_(v) 1.3a andcontains only Ca_(v) 1.2, the Ca_(v) 1.3a antagonists can be assayed forthe capacity to inhibit expression or activity of the Ca_(v) 1.2 calciumchannel relative to the Ca_(v) 1.3a channel.

In another embodiment, the antagonist of a Ca_(v) 1.3a calcium channelis an agent that inhibits a mammalian Ca_(v) 1.3a calcium channel.Preferably, the antagonist of the Ca_(v) 1.3a calcium channel is acompound that is, for example, a small organic molecule, naturalproduct, protein (e.g., antibody, peptide fragment), nucleic acid, orpeptidomimetic. Antagonists of Ca_(v) 1.3a calcium channels can beprepared and/or identified using suitable methods, such as the methodsdescribed herein or suitable modifications thereof.

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1-Preparation of Experimental Mouse Brain Slices

Brain slices of mice were prepared for use in different experimentalprocedures such as whole cell patch clamp and fluorescent stainingprocedures. Slices were obtained from 17-25 day old C57BL/6 mice(Harlan), Cav1.3^(−/−) mice (were re-derived from mice obtained fromJoerg Striessnig) or BAC D1/BAC D2 EGFP transgenic mice (obtained fromNathaniel Heintz), and P16-17 rats. All animals were handled in accordwith Northwestern University ACUC and NIH guidelines. Coronal slicescontaining the striatum or mesencephalon were prepared at a thickness of300-350 μm. Mice were prepared and sacrificed in one of two ways; 1) themice were anesthetized deeply with ketamine and xylaxine, transcardiallyperfused with oxygenated, ice-cold, artificial cerebral spinal fluid(ACSF) and decapitated, or 2) the mice or rats were deeply anesthetizedwith halothane and decapitated without perfusion. The brains wererapidly removed and sectioned in oxygenated, ice-cold, ACSF using aLeica VT1000S vibratome (Leica Microsystems). When the mice wereprepared and sacrificed using ketamine, the ACSF sectioning solutioncontained 124 mM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 26 mM NaHCO₃,1.2 mM NaH₂PO₄, and 10 mM D-(+)-glucose, while halothane prepared miceACSF sectioning solution contained 194 mM sucrose, 30 mM NaCl, 4.5 mMKCl, 1 mM MgCl₂, 26 mM NaHCO₃, 1.2 mM NaH₂PO₄ and 10 mM D-glucose. Thesolutions were periodically checked and adjusted to ensure theosmolarity stayed near 300 mOsm/l. Once sliced, the brain sections weretransferred to a holding chamber where they were completely submerged inACSF bubbled continuously with 95% O₂ and 5% CO₂ and maintained at roomtemperature (22°-23° C.) for at least 1 hour before using.

Example 2-Electrophysiology Procedures

Whole cell patch clamp techniques were used on tissue slices to evaluatethe effect of experimental conditions on the voltage gated calciumchannels. Whole cell voltage-clamp or current-clamp recordings wereperformed using standard techniques (Choi S and Lovinger D M, 1997,Proc. Natl. Acad. Sci 94:2665-2670; Day M et al., 2005, J. Neurosci.25:8776-87). Individual slices were transferred to a submersion stylerecording chamber and continuously superfused with ACSF at a rate of 2-3ml/min at 31-33° C. Whole cell voltage and current clamp recordings wereperformed on dopaminergic neurons detected in the slice with the help ofan infrared-differential interference contrast (IR-DIC) video microscopeupon which was mounted an Olympus OLY-150 camera/controller system(Olympus, Japan). For all experiments, experimental drugs weresuperfused with a temperature controlled system. Isradipine, nimodipine,and tetrodotoxin were made up as stock solutions and diluted immediatelybefore use. Patch electrodes were made by pulling TW150E-4 (WorldPrecisions Instruments, Sarasota Fla.) or BF150-86-10 glass on a P-97Flaming/Brown micropipette puller (Sutter Instrument Co.) and firepolished before recording. Pipette resistance was typically 2.5-6 MWafter filling with internal solution.

L-type calcium channels can be inhibited by dihydropyridine antagonists,such as nimodipine (FIG. 1) and isradipine (FIG. 4). Blockade withisradipine activates a homeostatic mechanism which involves adenylylcyclase and hyperpolarization activated cation channels, that restorespacemaking and dopaminergic neuron cell function through an alteredpacemaking mechanism. The sustained blockade of Ca_(v)1.3 channels leadsto re-emergence of the altered pacemaking mechanism that is dependentupon sodium and hyperpolarization activated cation channels. This showsthat blockade of Ca_(v)1.3 channels does not have significant sideeffects on brain dopaminergic function nor are obvious behavioralconsequences observed. As well, blockade of tetrodotoxin (TTX) sensitivesodium channels eliminates spikes but not the underlying oscillationsthat drive pacemaking.

Example 3-Procedure for Two Photon Laser Scanning Microscopy

Two photon laser scanning microscopy (2PLSM) was performed on mousetissue samples to visualize intracellular conditions. 2PLSM images ofmedium spiny neurons in 275 μm thick corticostriatal slices werevisualized with Alexa Fluor 594 (50 μM) by filling through the patchpipette. Following break in, the dye was loaded for at least 15 minutesprior to imaging. 2PLSM red signals (580-640 nm) were acquired using 810nm excitation with 90 MHz pulse repetition frequency and ˜250 fs pulseduration at the sample plane. Maximum projection images of the soma anddendritic field were acquired with a 60×/0.9NA water-dipping lens with0.27 μm² pixels and 2.6 μs pixel dwell time; ˜80 images were taken using0.7 μm focal increments. High magnification projections of dendriticsegments taken 50-100 μm from the soma were acquired with 0.17 μm²pixels and 10.2 μs dwell time and consisted of ˜20 images taken at 0.5μm focal steps. 2PLSM green signals (500-550 nm) were acquired fromneurons using 810 nm excitation.

The two-photon excitation source was a Chameleon-XR tunable laser system(705 nm to 980 nm) utilizing Ti:sapphire gain medium withall-solid-state active components and a computer optimized algorithm toensure reproducible excitation wavelength, average power, and peak power(Coherent Laser Group). Excitation at 810 nm with 90 MHz pulserepetition frequency and ˜250 fs pulse duration at the sample plane wasused for the two-photon excitation. Laser average power attenuation wasachieved with two Pockels cell electro-optic modulators (models 350-80and 350-50, Con Optics). The two cells were aligned in series to provideenhanced modulation range for fine control of the excitation dose (0.1%steps over four decades). Laser scanned images were acquired with aBio-Rad Radiance MPD system (Hemel Hempstead). Fluorescence emission wascollected by external or non-de-scanned photomultiplier tubes (PMT's).Green fluorescence (500 to 550 nm) was detected by a bialkali-cathodePMT and red Once fluorescence emission was collected, the systemdigitized the current from detected photons to 12 bits. The laser lighttransmitted through the sample was collected by the condenser lens andsent to another PMT to provide a bright-field transmission image inregistration with the fluorescent images. The stimulation, display, andanalysis software was a custom-written shareware package (WinFluor andPicViewer—John Dempster, Strathclyde University, Glasgow, Scotland; UK).

As can be seen in FIG. 3, pacemaking is accompanied by largefluctuations in calcium levels in dendrites of SNc dopaminergic neuronsthereby demonstrating that calcium levels are important in activity ofthese cells.

Example 4-Single Cell Reverse Transcription Polymerase Chain ReactionProcedure

Single cell reverse transcription polymerase chain reaction (scRT-PCR)was performed on cell nucleic acids to determine the levels of specificmRNA that a particular cell was producing. Neurons were acutelyisolated, harvested and profiled using protocols similar to thosepreviously described (Tkatch T et al., 2000, J. Neurosci 20:579-88).

As demonstrated in FIG. 2, the long splice variant of the Ca_(v)1.3gene, Ca_(v)1.3a, is present in SNc dopaminergic neurons and mediumspiny neurons.

Example 5-Behavioral Analysis Techniques Used to Evaluate Ca_(v)1.3Knock-Out Mice

Behavioral analysis was performed on Ca_(v)1.3 knock-out mice andcompared with the behavioral performance of wt mice in order todetermine if the deletion of the Ca_(v)1.3 subunits affected mousebehavioral phenotypes. Mice were evaluated using several differenttechniques; the pole test, the elevated plus maze (EPM), the cross maze,and a swimming version of the cross maze. Motor agility was assayedusing the pole test, anxiety related behavior and cognitive/learningbehaviors were evaluated using the cross maze, the swimming cross maze,and the EPM.

Test subjects were two cohorts of male mice (N=8 & 11). Each cohortcontained both Ca_(v)1.3 knock-out mice (N=9) and wt littermates (N=10).All of the mice were group housed. The mice were placed on a waterrestricted diet prior to cross maze testing so that they would bethirsty and work for water rewards. The mice received water duringtesting and for one hour at the end of the day when they had one hour offree access to water in their home cage. The weight, appearance andbehavior of the mice were monitored throughout the experiment in orderto insure that the mice were in healthy condition (all procedures wereapproved by Northwestern University's ACUC committee). The experimenterwas blind as to the genotype of the mice during data collection.

The pole test (Matsuura K, et al., 1997, J. Neurosci. Meth. 73:45-8) wasused to assess gross motor agility. Mice were placed at the top of an 8mm diameter pole with their head pointed up. The time taken for them todescend the 55 cm to the bottom of the pole was recorded. Resultsindicate that there was no significant difference between wt andknock-out mice for the time it took to invert themselves and descend thepole to the bottom.

The land based cross-maze was used to test the mice for motor andcognitive/learning abilities. The maze consisted of an elevated crossshaped platform with four white arms of equal length (35×6.5 cm) thatextended from a central area (6.5×6.5 cm) and which were enclosed byclear Plexiglas walls (15 cm tall). The maze was based on that describedby Middei S, et al., 2004, Behav. Brain Res. 154:527-34, except that thesidewalls extended the entire length of the maze, and the end of eacharm had a depression to contain a water reward (a 25 μl drop). Subjectsreceived five consecutive days of habituation sessions. During thesesessions, the mice were introduced into the maze at the south end, andthe east and west arms of the maze were blocked by Plexiglas barriers inorder not to induce a bias towards a particular east or west arm. Thedepression well at the end of the north arm contained 25 μl of water.Each mouse was given five trials per session with the opportunity toexplore the maze, discover the water in the depression at the end of thenorth arm, and learn to drink the water. The mice were removed from themaze 15 seconds after drinking. Mice that did not find the water withintwo minutes were guided to the water and then removed 15 seconds afterdrinking.

Experimental sessions began twice a day at the conclusion of thehabituation sessions. Mice were typically started from the distal end ofthe south arm and access to the north arm of the maze was blocked by aPlexiglas barrier so that the maze became a “T” maze. The maze was keptin a room with the same visual cues in place during each session. Themice were given five trials in which the north arm of the maze wasblocked. The goal arm (the east arm) contained 25 μl of water while thewest arm contained no water. The mice were introduced via the south armand allowed to choose an arm. Once the mice picked an arm, they wereenclosed in the arm for 15 seconds that was enough time for the mouse toreach the end of the arm, drink the water (if the correct arm waschosen) and scan the surroundings. No correction methods wereimplemented for incorrect arm choice. A mouse was considered to havereached the center of the maze when its front paws crossed the borderbetween the south arm and center region. The mice frequently exploredthe east or west arms with their nose and front paws, but an entry wasconsidered to have been made only after both hind paws and the snoutcrossed the border of the center region and a side arm.

Movement speed was calculated for traversing the cross maze. Micetypically went to the center area very quickly. The trial with theshortest latency for each session was analyzed using a repeated measuresANOVA. The results indicate no significant difference between the wt andknock-out mice. The mice spent much of their time in the center portionof the cross maze which suggested that they might be exhibiting anxietyrelated behaviors. The number of fecal boli deposited by each mouse onthe fifth day of training was recorded and analyzed for a differencebetween groups with a t-test. No significant difference was revealed.

A more rigorous examination of anxiety and exploration was performed bytesting the mice on the EPM (VideoMot2, TSE, Midland, Mich.) for asingle five minute session. The software calculated the time spent byeach mouse in the open and closed arms, as well as the number of entriesinto each type of arm. None of the variables measured exhibited asignificant difference between the wt and knock-out mice. The movementspeed during exploration in the EPM was measured and no significantdifference was found between genotypes.

Cognitive abilities were additionally assessed using the cross maze. Arepeated measures ANOVA of genotype by session for the percent ofcorrect choices indicated no significant difference between genotypes,but a significant increase from 49.5% correct on session 1 (50%indicates choosing randomly) to 83.2% correct on session 5 (ANOVA, F 4,68=14.1, P<0.0001).

As mentioned earlier, the mice in the cross maze quickly ran to thecenter and then often stayed in the center or traversed the south startarm. The latency between entering the center area and finally choosing aside arm to enter was analyzed using a repeated measures ANOVA for the12 sessions, and genotype as a factor. The results indicated nosignificant difference in this choice latency between the wt andknock-out mice. A detailed analysis of the exploratory activity prior tocommitting an entry to the east or west arm was done towards the end oftraining. Those results indicated no significant difference betweengenotypes in terms of how often the mice explored the east or west armwith nose pokes, but there was a significant preference (13.4 versus 5.5counts) for exploring the east, rewarded arm (F 1,9=22.0, P=0.001) whenthe two genotypes were considered together.

A probe trial was conducted after the initial five trials werecompleted. Mice were started from the far end of the north arm for thistrial and access to the south arm was blocked. The east and west armsboth contained 25 μl of water, and the subjects were allowed to pick andexplore an arm. The subjects were given a maximum of two minutes tochoose an arm during all trials. If this time limit was exceeded, thesubject was removed from the maze and given another trial. In order toremove any olfactory cue, the maze was wiped down with a sponge dampedwith water after each trial. Data were averaged for the two sessions perday prior to analysis. An analysis of the probe trials suggests thatknock-out mice were choosing which side to enter in a random fashionwhile the wt mice chose in a manner indicating a significant preferencefor hippocampally based spatial behavior rather than a striatal basedmotor response (F 1, 17=4.5, p=0.049), i.e., the wt mice continued toprefer the east (rewarded) arm even when started from the north insteadof the south arm. This difference between the knock-out and wt miceappeared to be dominated by the last session on day five since it wasthe only session to show a significant difference between the groupswhen analyzed with individual t-tests (t=2.1, df=17, p=0.049). Accordingto test results, the knock-out mice were responding in a random fashionand the wt mice were exhibiting a preference for a spatially basedbehavior which is expected for C57Bl/6 mice (Middei S, et al., 2004,Behav. Brain Res. 154:527-34). These data could be interpreted to meanthat wt C57BL6 mice are dominated by hippocampal based spatial behaviorand that the striatal system of Ca_(v)1.3 knock-out mice are more ableto influence motor behavior.

The behavioral analysis of the land based cross maze was complicated bythe tendency of the mice to stop ambulating during a trial. Therefore, awater based version was created and used to determine if any differencesbetween genotypes were present. The mice swam well and did not stop andfloat during the test. The maze was made of clear acrylic and had armsthat extended 77.5 cm from one end to the other. The alley ways were 15cm wide with 17.5 cm high walls. The escape platform was 6×6 cm and waselevated 10.5 cm from the base of the maze. There were no edges for themouse to grab and the alleys were wide enough that the mouse could notbrace itself between the walls. The maze was submerged within a pool of25° C. water that was made opaque with white tempera paint. HVS Imagesoftware was used to collect latency and path length data. A repeatedmeasures ANOVA for the percent of correct choices across 10 trainingsessions indicated significant improvement in choosing the correct armwith no significant difference between genotype, and no significantinteraction of group and learning. The percent of correct responsesincreased from a minimum of 60% correct on the second session (50% israndom) to a maximum of 100% correct on the eighth session. The meanlatency to find the hidden escape platform in the rewarded arm of themaze was also measured and analyzed. This escape latency decreasedsignificantly during the first five sessions from a maximum of 13.5seconds on session two to a minimum of 4.0 seconds on session five.There was no significant difference between the wt and knock-out mice,and no interaction of group and training. The same results were foundwhen the distance traveled to the goal was analyzed.

The mice were put back into the water cross maze two days later andreleased to swim to a visible platform (instead of a hidden platform)that was located in the arm opposite that which had been rewarded withthe hidden platform. This test is considered sensitive to striatalinvolvement since striatal based habit learning might impair the abilityof the mouse to execute a new response pattern. The results indicatedimprovement during the first session, but there was no significantdifference between the wt and knock-out mice for the latency to climbonto the escape platform during any of the three days of testing. Thefirst trial of the visible testing is interesting and suggests that theknock-out mice have more “cognitive flexibility” than the wt mice (i.e.the hippocampus can overcome the striatum). Finally, the latency for themice to reach the escape platform in the water cross maze during thelast five days of testing was analyzed with a repeated measures ANOVA.The results indicated no significant difference between the groups.

As described above, when Ca_(v)1.3 knock-out mice were compared withlitter mate controls for differences in motor behavior, agility,anxiety, learning, and preference for spatial or response basedambulation no differences due to genotype were noted, except that therewas a hint of a preference for spatially guided ambulation in wt miceand a random probability that a knock-out mouse would use either spatialguidance or response guidance. Therefore, the elimination of theCa_(v)1.3 calcium channel, thereby shifting the pacemaking mechanism tothe more juvenile form of pacemaking that shifts dopaminergic neurons tosodium channel dependent pacemaking, does not lead to any obviousbehavioral consequences.

Example 6-siRNA Methods for Inhibition of Ca_(v)1.3 Expression in SNcDopaminergic Neurons

Another way of inhibiting Ca_(v) 1.3 channels in SNc dopaminergicneurons is to inhibit translation of the Ca_(v) 1.3 mRNA using siRNAtechnique. The target sequences for the siRNAs are selected to avoidpotential inhibition of other calcium channels. For example, conservedregions, like transmembrane domains and the proline rich region, areavoided. The greatest sequence diversity in calcium channel subunits isfound in the 3′ region of the mRNA that codes for the cytoplasmiccarboxy terminal region. This segment of the mRNA is preferablytargeted. BLAST sequence analysis programs can be used to screencandidate siRNA for specificity. Efficiency and specificity of thecalcium channel inhibition can be assessed using real-time quantitativePCR and Western blotting. In some embodiments, viral constructs thatexpress small hairpin RNA (shRNA) are used for administration.

Example 7

Analysis of Rasagiline and Isradipine on Brain Function in Mice

This Example describes an analysis of brain function in miceadministered rasagiline, isradipine, and the combination thereof.

Materials and Methods

Brain Slice Preparation

Slices were obtained from 25-35 day old C57BL/6 mice (Charles River) orfrom described transgenic mice (mito-roGFP). Mice were anesthetized withkatamine/xylazine mixture, followed by a transcardial perfusion withice-cold oxygenated artificial cerebrospinal fluid (ACSF) containing thefollowing (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl, 1MgCl, and 25 dextrose, pH 7.3, osm 315-320 mOsm/L. After perfusion, micewere decapitated and brains were rapidly removed and sectioned inice-cold oxygenated ACSF using a vibratome (VT 1000S; LeicaMicrosystems). Midbrain slices (220 μm) were recovered in ACSF at 34° C.for 30 minutes before experiments were started (electrophysiology,calcium and roGFP imaging). The external ACSF solutions were bubbledwith 95% O2/5% CO2 at all times to maintain oxygenation and a pH≈7.4.

Electrophysiology

Whole-cell current-clamp recordings were done using standard techniques.Slices were transferred to a submersion-style recording chamber mountedon an Olympus BX51-WIF upright, fixed-stage microscope (Melville, N.Y.).The slices where continuously perfused with ≈1.5 ml/min ACSF at (34-35°C.). For patching, Substantia nigra pars compacta neurons (SNc) werevisually identified by both topology and morphology. 60×/0.9 NAwater-dipping objective was used for patching the cell somas, 50-100 umbelow slice surface, and subsequent line scan fluorescence recordings.Patch electrodes were made by pulling BF150-86-10 glass on a P-97Flaming/Brown micropipette puller (both from Sutter Instrument Co.,Novato, Calif.). The pipette solution contained the following (in mM):135 KMeSO₄ (ICN Biomedicals Inc., Aurora, Ohio), 5 KCl, 0.5 CaCl₂, 5HEPES, 2 MgATP, 0.2 Na₂GTP, pH=7.25-7.3 with KOH, 270 mOsm/l. Asmeasured in the bath, the pipette resistance was ˜4 MΩ. Seals wereformed in voltage-clamp mode on the cell somas with series resistance >1GΩ. After rupture in whole-cell configuration, series-resistancedecreased to 10-15 MΩ. Electrophysiological recordings were obtainedwith a Multiclamp 700B amplifier (Axon Instruments, Union City, Calif.)and then digitized inside the scanning computer (PCI MIO-16E-4, NationalInstruments, Austin, Tex.). The stimulation, display, and analysissoftware was a custom-written shareware package, WinFluor (JohnDempster, Strathclyde University, Glasgow, Scotland; UK). WinFluorautomated and synchronized the two-photon excited fluorescence with theelectrophysiological stimulation.

Photometry-PLSM Ca2+ Imaging

For dendritic Ca2+ measurements, SNc neurons were loaded with internalsolutions supplemented with Alexa Fluor 594 (20 μM) and Fluo-4 (50 μM)through the patch pipette. All experiments were performed at 32-34° C.Imaging took place after 15-20 min of dye loading. Images were acquiredwith an Olympus LUMPFL 60×/1.0 NA water-dipping objective lens. Thetwo-photon excitation source was a Chameleon-ultral laser system (690 to1040 nm) from Coherent Laser Group. Optical signals were acquired using820 nm excitation (80-MHz pulse repetition frequency and ˜250 fs pulseduration) to simultaneously excite Alexa and Fluo-4 dyes. Laser powerattenuation was achieved with two Pockels' cell electro-optic modulators(models M350-80-02-BK and M350-50-02-BK; Con Optics) controlled byPrairieView and WinFluor, respectively. The two cells are aligned inseries to provide: enhanced modulation range for fine control of theexcitation dose (0.1% steps over five decades), to limit maximum power,and to serve as a rapid shutter during line scan acquisitions. Thelaser-scanned images were acquired with a Prairie Technologies Ultimasystem. The fluorescence emission was collected by non-de-scannedphotomultiplier tubes (PMTs). The green (490-560 nm) and red (580-630nm) fluorescence were each detected by a multi-alkali-cathode PMT. Thesystem digitized the current from detected photons to 12 bits. The laserlight transmitted through the sample was collected by the condenser lensand sent through a Dodt contrast tube (Luigs and Neumann) to another PMT(R3896, Hamamatsu) to provide a bright-field transmission image (PrairieUSB transmission detector) in registration with the fluorescent images.Fluorescence measurements were taken in a sample plane along dendriticsegments (80-100 μm from the soma). Line scan signals were acquired at 6ms per line and 512 pixels per line with 0.18 μm pixels and 10 μs pixeldwell time.

Mito-roGFP Imaging

Optical imaging of roGFP signals was acquired using a 920 nm excitationbeam (80 MHz pulse repetition frequency and 250 fs pulse duration), in afixed plane of focus with a pixel size of 0.18 μm and a 10-12 μs pixeldwell time. Fifteen to twenty seconds of roGFP (150-300 frames)dendritic signal was collected per trial. Records with drifting baseline(due to photobleaching or photooxidation of roGFP) were discarded. Atthe end of all experiments, the maximum and minimum fluorescence ofmito-roGFP were determined by application of 2 mM DTT to fully reducethe mitochondria and then 100 μM Aldrithiol (Ald) to fully oxidize themitochondria. The relative oxidation was calculated as1-[(F-FAld)/(FDTT-FAld)].

Chronic Drug Treatment

In all of the experiments shown, rasagiline and isradipine weredelivered separately by subcutaneous Alzet osmotic minipumps (model1002; Alzet, Cupertino, Calif.). One group of animals was implanted withsingle pump of isradipine or rasagiline and other group was implantedwith 2 pumps. Isradipine was dissolved in vehicle (DMSO/PEG300/water) atfollowing concentrations: 0.03, 0.3 and 3 mg/kg/day, and rasagiline wasdissolved in saline at concentrations of 1 and 3 mg/kg/day. Theseconcentrations were used individually or as combinations in differentgroups of animals (see results). Isradipine and rasagiline werecontinuously delivered with a flow rate of 0.25 ml/hr for 10-14 days.Prior implantation, pumps were placed in 0.9% saline overnight at 37° C.to insure immediate release of the drug. Pumps were implantedsubcutaneously following manufacturer's guidelines, as follow. Undershort-term anesthesia, an incision was made between scapulas and asubcutaneous pocket was formed via blunt dissection. The infusion end ofthe pump was placed away from the site of incision, which was closedwith wound clips.

Data Analysis

Data were visualized and analyzed with custom image-processing sharewaresoftware. PicViewer and WinFluor (John Dempster), and IGOR v4.05(WaveMetrics, Lake Oswego, Oreg.) were employed for data smoothing andstatistics. Pooled data are graphically presented either asmeans±standard error (SE), or as box plots. In the box plots the medianvalue of the data is represented by the central bar. The outer edges ofthe box split the upper and lower halves of the data in half again(interquartile range). Finally, the whiskers delineate the upper andlower outerquartiles of the data (Systat v5.2, Evanston, Ill.).

Results

The results of this Example are shown in FIGS. 6-9. FIG. 6 shows thatrasagiline does not alter pacemaking rate or intracellular calciumoscillations associated with pacemaking. Left: image of an SNc DA neuronin an ex vivo brain slice with an annulus drawn to show the area fromwhich dendritic calcium measurements were made. Middle: Whole cellvoltage measurements from an SNc DA neuron in a control slice; bottomare calcium oscillations associated with pacemaking made using theindicator Fluo4 and 2PLSM. Right: Similar measurements from a brainslice taken following rasagiline treatment. FIG. 7 shows thatmitochondrial oxidant stress is diminished by chronic isradipine orchronic rasagiline treatment. In FIG. 7a , the top panels shows aphotomicrograph of mesencephalon of BAC-mito-roGFP mouse showingtransgene expression in SNc and VTA DA neurons, while the bottom panelsshows an image of a single neuron. FIG. 7b shows redox measurements inex vivo brain slices were calibrated by application of strong reducing(DTT) and oxidizing agents (aldrithiol) at the end of each experiment.FIG. 7c shows representative control and acute rasagiline-inducedresponses. FIG. 7d shows box plots summarizing mitochondrial oxidantstress measurements from mice given isradipine or rasagiline acutely orchronically (1 week) with s.c. osmotic minipumps. N>4 in each group.FIG. 8 shows that the effects of chronic administration of isradipine orrasagiline were dose-dependent. FIG. 8a shows box plots summarizingmitochondrial oxidant stress measurements in SNc DA neurons in ex vivobrain slices from mice given saline or isradipine. Acute treatment atthe time of measurement (1 μM); other drugs were given for 1 week vias.c. osmotic minipump. FIG. 8b shows the same as in a, but withrasagiline. Acute rasagiline was given at X μM. N>4 in each group. FIG.9 shows that the combination of low dose isradipine and rasagiline ismore effective than either alone. FIG. 9a shows a mouse with two s.c.osmotic minipumps; drugs were given for 1 week prior to sacrifice. FIG.9b shows box plots summarizing mitochondrial oxidant stress measurementsin SNc DA neurons in ex vivo brain slices from mice given drugs alone orin combination. N>4 in each group.

This Example showed that rasagiline has no effect on pacemaking of SNcdopaminergic neurons or on calcium entry linked to pacemaking. Acuterasagiline treatment lowers mitochondrial oxidant stress in SNcdopaminergic neurons. Chronic treatment (>1 wk) further lowers stress.The combination of low dose isradipine and rasagiline more effectivelylowers mitochondrial stress than either treatment alone.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

We claim:
 1. A method of treatment for dopaminergic disorderscomprising: administering to a subject having a dopaminergic disorder:i) a first compound that inhibits a voltage-gated calcium channel of thetype Ca_(v)1.3, and ii) a second compound that is monoamine oxidaseinhibitor and/or is a nitric oxide synthase inhibitor.
 2. The method ofclaim 1, wherein said first compound comprises a dihydropyridine calciumchannel blocker.
 3. The method of claim 1, wherein said first compoundcomprises isradipine and said second compound comprises rasagiline or arasagiline derivative.
 4. The method of claim 1, wherein saidadministering said first and second compounds to said subject isrepeated on multiple days.
 5. The method of claim 4, wherein no morethan 9 mg of said first compound is administered to said subject on anygiven day.
 6. The method of claim 5, wherein said first compoundcomprises isradipine.
 7. The method of claim 5, wherein said secondcompound comprises rasagiline or a rasagiline derivative.
 8. The methodof claim 4, wherein said first compound comprises isradipine, whereinsaid second compound comprises rasagiline or a rasagiline derivative,wherein between about 0.5 mg and about 5.0 mg of said isradipine isadministered per day, and wherein about 0.5 mg and about 2.0 mg of saidrasagiline or rasagiline derivative is administered per day.
 9. Themethod of claim 1, wherein said first compound is in a controlledrelease formulation.
 10. A composition comprising: i) a first compoundthat inhibits a voltage-gated calcium channel of the type Ca_(v)1.3, andii) a second compound that is monoamine oxidase inhibitor and/or is anitric oxide synthase inhibitor.
 11. The composition of claim 10,wherein said first compound comprises a dihydropyridine calcium channelblocker.
 12. The composition of claim 10, wherein said first compoundcomprises isradipine and said second compound comprises rasagiline or arasagiline derivative.
 13. The composition of claim 10, wherein saidfirst composition contains no more than 9 mg of said first compound. 14.The composition of claim 13, wherein said first compound comprisesisradipine.
 15. The composition of claim 13, wherein said secondcompound comprises rasagiline or a rasagiline derivative.
 16. Thecomposition of claim 10, wherein said first compound comprisesisradipine, wherein said second compound comprises rasagiline or arasagiline derivative, wherein said composition contains between about0.5 mg and about 5.0 mg of said isradipine and between about 0.5 mg andabout 2.0 mg of said rasagiline or rasagiline derivative.
 17. Thecomposition of claim 10, wherein said first compound is in a controlledrelease formulation within said composition.
 18. A kit comprising: i) afirst composition comprising a first compound that inhibits avoltage-gated calcium channel of the type Ca_(v)1.3, and ii) a secondcomposition comprising a second compound that is monoamine oxidaseinhibitor and/or is a nitric oxide synthase inhibitor.
 19. The kit ofclaim 18, wherein said first compound comprises isradipine and saidsecond compound comprises rasagiline or a rasagiline derivative.
 20. Thekit of claim 18, wherein said first composition contains no more than 9mg of said first compound.